Major haplotype divergence including multiple germin-like protein genes, at the wheat Sr2 adult plant stem rust resistance locus

Background The adult plant stem rust resistance gene Sr2 was introgressed into hexaploid wheat cultivar (cv) Marquis from tetraploid emmer wheat cv Yaroslav, to generate stem rust resistant cv Hope in the 1920s. Subsequently, Sr2 has been widely deployed and has provided durable partial resistance to all known races of Puccinia graminis f. sp. tritici. This report describes the physical map of the Sr2-carrying region on the short arm of chromosome 3B of cv Hope and compares the Hope haplotype with non-Sr2 wheat cv Chinese Spring. Results Sr2 was located to a region of 867 kb on chromosome 3B in Hope, which corresponded to a region of 567 kb in Chinese Spring. The Hope Sr2 region carried 34 putative genes but only 17 were annotated in the comparable region of Chinese Spring. The two haplotypes differed by extensive DNA sequence polymorphisms between flanking markers as well as by a major insertion/deletion event including ten Germin-Like Protein (GLP) genes in Hope that were absent in Chinese Spring. Haplotype analysis of a limited number of wheat genotypes of interest showed that all wheat genotypes carrying Sr2 possessed the GLP cluster; while, of those lacking Sr2, some, including Marquis, possessed the cluster, while some lacked it. Thus, this region represents a common presence-absence polymorphism in wheat, with presence of the cluster not correlated with presence of Sr2. Comparison of Hope and Marquis GLP genes on 3BS found no polymorphisms in the coding regions of the ten genes but several SNPs in the shared promoter of one divergently transcribed GLP gene pair and a single SNP downstream of the transcribed region of a second GLP. Conclusion Physical mapping and sequence comparison showed major haplotype divergence at the Sr2 locus between Hope and Chinese Spring. Candidate genes within the Sr2 region of Hope are being evaluated for the ability to confer stem rust resistance. Based on the detailed mapping and sequencing of the locus, we predict that Sr2 does not belong to the NB-LRR gene family and is not related to previously cloned, race non-specific rust resistance genes Lr34 and Yr36

Major haplotype divergence including multiple germin-like protein genes, at the wheat Sr2 adult plant stem rust resistance locus

Background The adult plant stem rust resistance gene Sr2 was introgressed into hexaploid wheat cultivar (cv) Marquis from tetraploid emmer wheat cv Yaroslav, to generate stem rust resistant cv Hope in the 1920s. Subsequently, Sr2 has been widely deployed and has provided durable partial resistance to all known races of Puccinia graminis f. sp. tritici. This report describes the physical map of the Sr2-carrying region on the short arm of chromosome 3B of cv Hope and compares the Hope haplotype with non-Sr2 wheat cv Chinese Spring. Results Sr2 was located to a region of 867 kb on chromosome 3B in Hope, which corresponded to a region of 567 kb in Chinese Spring. The Hope Sr2 region carried 34 putative genes but only 17 were annotated in the comparable region of Chinese Spring. The two haplotypes differed by extensive DNA sequence polymorphisms between flanking markers as well as by a major insertion/deletion event including ten Germin-Like Protein (GLP) genes in Hope that were absent in Chinese Spring. Haplotype analysis of a limited number of wheat genotypes of interest showed that all wheat genotypes carrying Sr2 possessed the GLP cluster; while, of those lacking Sr2, some, including Marquis, possessed the cluster, while some lacked it. Thus, this region represents a common presence-absence polymorphism in wheat, with presence of the cluster not correlated with presence of Sr2. Comparison of Hope and Marquis GLP genes on 3BS found no polymorphisms in the coding regions of the ten genes but several SNPs in the shared promoter of one divergently transcribed GLP gene pair and a single SNP downstream of the transcribed region of a second GLP. Conclusion Physical mapping and sequence comparison showed major haplotype divergence at the Sr2 locus between Hope and Chinese Spring. Candidate genes within the Sr2 region of Hope are being evaluated for the ability to confer stem rust resistance. Based on the detailed mapping and sequencing of the locus, we predict that Sr2 does not belong to the NB-LRR gene family and is not related to previously cloned, race non-specific rust resistance genes Lr34 and Yr36

An efficient approach to BAC based assembly of complex genomes

Background: There has been an exponential growth in the number of genome sequencing projects since the introduction of next generation DNA sequencing technologies. Genome projects have increasingly involved assembly of whole genome data which produces inferior assemblies compared to traditional Sanger sequencing of genomic fragments cloned into bacterial artificial chromosomes (BACs). While whole genome shotgun sequencing using next generation sequencing (NGS) is relatively fast and inexpensive, this method is extremely challenging for highly complex genomes, where polyploidy or high repeat content confounds accurate assembly, or where a highly accurate ‘gold’ reference is required. Several attempts have been made to improve genome sequencing approaches by incorporating NGS methods, to variable success. Results: We present the application of a novel BAC sequencing approach which combines indexed pools of BACs, Illumina paired read sequencing, a sequence assembler specifically designed for complex BAC assembly, and a custom bioinformatics pipeline. We demonstrate this method by sequencing and assembling BAC cloned fragments from bread wheat and sugarcane genomes. Conclusions: We demonstrate that our assembly approach is accurate, robust, cost effective and scalable, with applications for complete genome sequencing in large and complex genomes

The improved assembly of 7DL chromosome provides insight into the structure and evolution of bread wheat

Wheat is one of the most important staple crops worldwide and also an excellent model species for crop evolution and polyploidization studies. The breakthrough of sequencing the bread wheat genome and progenitor genomes lays the foundation to decipher the complexity of wheat origin and evolutionary process as well as the genetic consequences of polyploidization. In this study, we sequenced 3286 BACs from chromosome 7DL of bread wheat cv. Chinese Spring and integrated the unmapped contigs from IWGSC v1 and available PacBio sequences to close gaps present in the 7DL assembly. In total, 8043 out of 12 825 gaps, representing 3 491 264 bp, were closed. We then used the improved assembly of 7DL to perform comparative genomic analysis of bread wheat (Ta7DL) and its D donor, Aegilops tauschii (At7DL), to identify domestication signatures. Results showed a strong syntenic relationship between Ta7DL and At7DL, although some small rearrangements were detected at the distal regions. A total of 53 genes appear to be lost genes during wheat polyploidization, with 23% (12 genes) as RGA (disease resistance gene analogue). Furthermore, 86 positively selected genes (PSGs) were identified, considered to be domestication‐related candidates. Finally, overlapping of QTLs obtained from GWAS analysis and PSGs indicated that TraesCS7D02G321000 may be one of the domestication genes involved in grain morphology. This study provides comparative information on the sequence, structure and organization between bread wheat and Ae. tauschii from the perspective of the 7DL chromosome, which contribute to better understanding of the evolution of wheat, and supports wheat crop improvement

Sequence Composition and Gene Content of the Short Arm of Rye (Secale cereale) Chromosome 1

BACKGROUND: The purpose of the study is to elucidate the sequence composition of the short arm of rye chromosome 1 (Secale cereale) with special focus on its gene content, because this portion of the rye genome is an integrated part of several hundreds of bread wheat varieties worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Multiple Displacement Amplification of 1RS DNA, obtained from flow sorted 1RS chromosomes, using 1RS ditelosomic wheat-rye addition line, and subsequent Roche 454FLX sequencing of this DNA yielded 195,313,589 bp sequence information. This quantity of sequence information resulted in 0.43× sequence coverage of the 1RS chromosome arm, permitting the identification of genes with estimated probability of 95%. A detailed analysis revealed that more than 5% of the 1RS sequence consisted of gene space, identifying at least 3,121 gene loci representing 1,882 different gene functions. Repetitive elements comprised about 72% of the 1RS sequence, Gypsy/Sabrina (13.3%) being the most abundant. More than four thousand simple sequence repeat (SSR) sites mostly located in gene related sequence reads were identified for possible marker development. The existence of chloroplast insertions in 1RS has been verified by identifying chimeric chloroplast-genomic sequence reads. Synteny analysis of 1RS to the full genomes of Oryza sativa and Brachypodium distachyon revealed that about half of the genes of 1RS correspond to the distal end of the short arm of rice chromosome 5 and the proximal region of the long arm of Brachypodium distachyon chromosome 2. Comparison of the gene content of 1RS to 1HS barley chromosome arm revealed high conservation of genes related to chromosome 5 of rice. CONCLUSIONS: The present study revealed the gene content and potential gene functions on this chromosome arm and demonstrated numerous sequence elements like SSRs and gene-related sequences, which can be utilised for future research as well as in breeding of wheat and rye

A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor

The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions

A chromosome conformation capture ordered sequence of the barley genome

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Stem rust resistance in wheat is suppressed by a subunit of the mediator complex

Stem rust is an important disease of wheat that can be controlled using resistance genes. The gene SuSr-D1 identified in cultivar 'Canthatch' suppresses stem rust resistance. SuSr-D1 mutants are resistant to several races of stem rust that are virulent on wild-type plants. Here we identify SuSr-D1 by sequencing flow-sorted chromosomes, mutagenesis, and map-based cloning. The gene encodes Med15, a subunit of the Mediator Complex, a conserved protein complex in eukaryotes that regulates expression of protein-coding genes. Nonsense mutations in Med15b.D result in expression of stem rust resistance. Time-course RNAseq analysis show a significant reduction or complete loss of differential gene expression at 24h post inoculation in med15b.D mutants, suggesting that transcriptional reprogramming at this time point is not required for immunity to stem rust. Suppression is a common phenomenon and this study provides novel insight into suppression of rust resistance in wheat. Stem rust is an important disease of wheat and resistance present in some cultivars can be suppressed by the SuSr-D1 locus. Here the authors show that SuSr-D1 encodes a subunit of the Mediator Complex and that nonsense mutations are sufficient to abolish suppression and confer stem rust resistance

Shifting the limits in wheat research and breeding using a fully annotated reference genome

Introduction: Wheat (Triticum aestivum L.) is the most widely cultivated crop on Earth, contributing about a fifth of the total calories consumed by humans. Consequently, wheat yields and production affect the global economy, and failed harvests can lead to social unrest. Breeders continuously strive to develop improved varieties by fine-tuning genetically complex yield and end-use quality parameters while maintaining stable yields and adapting the crop to regionally specific biotic and abiotic stresses. Rationale: Breeding efforts are limited by insufficient knowledge and understanding of wheat biology and the molecular basis of central agronomic traits. To meet the demands of human population growth, there is an urgent need for wheat research and breeding to accelerate genetic gain as well as to increase and protect wheat yield and quality traits. In other plant and animal species, access to a fully annotated and ordered genome sequence, including regulatory sequences and genome-diversity information, has promoted the development of systematic and more time-efficient approaches for the selection and understanding of important traits. Wheat has lagged behind, primarily owing to the challenges of assembling a genome that is more than five times as large as the human genome, polyploid, and complex, containing more than 85% repetitive DNA. To provide a foundation for improvement through molecular breeding, in 2005, the International Wheat Genome Sequencing Consortium set out to deliver a high-quality annotated reference genome sequence of bread wheat. Results: An annotated reference sequence representing the hexaploid bread wheat genome in the form of 21 chromosome-like sequence assemblies has now been delivered, giving access to 107,891 high-confidence genes, including their genomic context of regulatory sequences. This assembly enabled the discovery of tissue- and developmental stage–related gene coexpression networks using a transcriptome atlas representing all stages of wheat development. The dynamics of change in complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. Aspects of the future value of the annotated assembly for molecular breeding and research were exemplarily illustrated by resolving the genetic basis of a quantitative trait locus conferring resistance to abiotic stress and insect damage as well as by serving as the basis for genome editing of the flowering-time trait. Conclusion: This annotated reference sequence of wheat is a resource that can now drive disruptive innovation in wheat improvement, as this community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding. Importantly, the bioinformatics capacity developed for model-organism genomes will facilitate a better understanding of the wheat genome as a result of the high-quality chromosome-based genome assembly. By necessity, breeders work with the genome at the whole chromosome level, as each new cross involves the modification of genome-wide gene networks that control the expression of complex traits such as yield. With the annotated and ordered reference genome sequence in place, researchers and breeders can now easily access sequence-level information to precisely define the necessary changes in the genomes for breeding programs. This will be realized through the implementation of new DNA marker platforms and targeted breeding technologies, including genome editing